Table 1.
Functional/Cross Reactive Antibodies.
Figure 1.
Markers to identify cells of epithelial origin.
Identification of intestinal cells of epithelial origin within the porcine small intestine and colon are shown. Immunostaining for EpCAM, a pan-epithelial transmembrane protein, demonstrated expression in the basolateral membrane of all cells in both the small intestine and colon arranged along the luminal monolayer of the epithelial mucosa. Immunostaining for Villin, a protein associated with the microvillar actin filaments, showed a gradient of expression localized to the apical border of small intestinal and colonic epithelial cells with increasing intensity in cells located closer to the lumen. All specific markers (red). Nuclei (blue). Scale bar 200 µm, inset scale bar 50 µm.
Figure 2.
Markers to assess proliferation and apoptosis.
Identification of proliferative cells in different stages of the cell cycle and those undergoing apoptosis in porcine small intestine and colon are shown. All proliferative markers localized to the nuclei of positive epithelial cells. Immunostaining for PCNA, a general marker for cellular proliferation, demonstrated the greatest number of positive cells compared to the other markers of proliferation. Immunostaining for MCM2, a marker of cells at the G1 stage of the cell cycle, was localized to a subpopulation of cells within the crypt base. Immunostaining for BrdU, a marker of cells within the S stage of the cell cycle, was also localized to a subpopulation of cells within the crypt base. Immunostaining for pH3, a marker for cells between the G2 – M stage of the cell cycle was similarly localized but to fewer cells. Immunostaining for cleaved caspase 3, an indicator of apoptosis, marked a few expressing cells near the villus tip within small intestine and the luminal surface of colon. All specific markers (red). Nuclei (blue) Scale bar 200 µm, inset scale bar 50 µm.
Figure 3.
Markers to identify stem, progenitor, and transit amplifying cells.
Identification of stem, progenitor, and transit amplifying cells in porcine small intestine and colon are shown. Immunostaining for SOX9, a member of the SRY-family of transcription factors that is primarily expressed in CBC cells and transit-amplifying progenitor cells, is localized to the nuclei of all cells within the crypt base of both the small intestine and colon. Immunostaining for SOX9 demonstrates colocalization with the proliferative cells (PCNA+) at the crypt base. Immunostaining for HOPX, an atypical homeodomain containing protein, demonstrated marking of cells consistent in location and numbers with a ‘reserve’ IESC population. All specific markers (red or green). Nuclei (blue). Scale bar 200 µm, inset scale bar 50 µm.
Table 2.
Non Functional Antibodies.
Figure 4.
Markers to identify the absorptive cell lineage.
Identification of absorptive enterocytes in porcine small intestine and colon are shown. Immunostaining for enterocytes demonstrates sucrase isomaltase, a digestive enzyme, marking the apical brush- border of cells in the small intestine. Carbonic anhydrase, a digestive enzyme, positively identified absorptive cells in the colon with staining localized to the apical brush- border of these cells. All specific markers (red). Nuclei (blue). Scale bar 200 µm, inset scale bar 50 µm.
Figure 5.
Markers to identify the secretory cell lineage.
Identification of secretory cells in porcine small intestine and colon are shown. Immunostaining for enteroendocrine cells, the hormone secreting cells important to gut homeostasis, using antibodies against CgA, SST, GLP-1 and GAST, demonstrated staining within the cytoplasm of positive cells of the small intestine. A similar pattern of staining was identified in the colon for CgA, SST and GLP-1. All specific markers (red). Nuclei (blue). Scale bar 200 µm, inset scale bar 50 µm.
Figure 6.
Markers to identify goblet cells.
Identification of goblet cells in porcine small intestine and colon are shown. Goblet cells, the mucus producing cells, were identified by immunostaining for MUC2, a component of mucin, and UEA-1, a lectin that specifically binds to alpha-linked fructose receptors. The mucinous secretions of multiple cells in both the small intestine and colon were positively identified. All specific markers (red). Nuclei (blue). Scale bar 200 µm, inset scale bar 50 µm.
Figure 7.
Transmission electron microscopic epithelial characterization.
The ultrastructural appearance and characterization of porcine small intestine and colonic epithelial cells are shown. All cell types were morphologically distinguishable. Crypt base columnar stem cells, goblet cells, enteroendocrine cells, and absorptive enterocytes are all marked with asterisks. ‘L’ indicates lumen. Small Intestinal images, scale bar 2 µm. Colon images, scale bar 5 µm.
Table 3.
Primers designed for gene expression in porcine intestine.
Figure 8.
In vitro culture of porcine crypts.
The in vitro isolation, growth and maintenance of enteroids derived from porcine intestinal crypts are shown. On the day of collection (day 0) crypts maintain their morphologic appearance. As the enteroids develop they become enterospheres (day 2) and progressively enlarge and form complex structures with a pseudolumen and crypt-like structures (days 4, 8, 14, 21). Scale Bar 100 µm.
Figure 9.
Markers to identify cell lineages within in vitro cultures.
The identification of specific cell lineages within in vitro cultures of porcine crypts is shown. The existence of stem/progenitor and differentiated lineages were confirmed in enteroids utilizing the established genetic biomarkers for cell lineage identification: anti-SOX9 (stem/progenitor), anti-PCNA (proliferation), anti-CgA (enteroendocrine), anti-MUC2 (goblet) and anti-sucrase isomaltase (absorptive enterocyte) antibodies. All specific markers (red). Nuclei, blue. Scale Bar 50 µm, inset scale bar 10 µm.